In-situ'tumor capture 'type electrostatic spinning patch as well as preparation method and application thereof

Abstract

The invention discloses an in-situ'tumor capture 'type electrostatic spinning patch as well as a preparation method and application thereof. The invention relates to an in-situ'tumor capture 'type electrostatic spinning patch for sequential treatment in and after pancreatic cancer resection. The electrostatic spinning patch comprises a gelatin / hyaluronic acid-based hemostatic sponge layer with a capture function, a gemcitabine slow-release layer and a polycaprolactone backing layer. According to the invention, the dual core requirements of hemostasis and cancer resistance during operation and after operation are integrated, and a long-acting protective barrier is constructed at the local part of the operation through continuous drug delivery. Therefore, the efficiency of removing residual lesions is remarkably improved, local recurrence and distant metastasis of tumors can be effectively prevented, and finally a safer, more lasting and more comprehensive comprehensive treatment new strategy is provided for pancreatic cancer postoperative patients.

Description

Technical Field

[0001] This invention belongs to the medical field, and particularly relates to an in-situ "tumor capture" type electrospun patch for sequential treatment during and after pancreatic cancer resection, its preparation method, and its application. Background Technology

[0002] Pancreatic cancer (PC) is a highly malignant gastrointestinal tumor characterized by its insidious nature and aggressiveness, resulting in a very poor overall prognosis, hence its nickname "the king of cancers." According to multiple global clinical studies, the overall five-year survival rate for PC patients is less than 10%, and the incidence and mortality rates of pancreatic cancer are showing an increasing trend year by year (Am J Gastroenterol. 2007 Dec;102(12):2696-2707). Based on domestic and international guidelines for the diagnosis and treatment of pancreatic cancer, surgical resection remains the primary means of rapidly controlling the progression of pancreatic cancer and achieving clinical cure. However, due to the high rates of postoperative recurrence and distant metastasis, the long-term prognosis for patients remains unsatisfactory, with a persistently high postoperative mortality rate. Studies show that even in patients who undergo complete microscopic tumor resection, the five-year survival rate is still less than 25%, with more than half experiencing local recurrence or distant metastasis within two years post-surgery (J Am Coll Surg. 1999 Jul;189(1):1-7).

[0003] Minimal residual lesions at the surgical resection margin are a significant cause of tumor recurrence. These minute lesions are often difficult to detect intraoperatively by the naked eye or imaging techniques. Furthermore, substantial evidence suggests that unavoidable intraoperative damage to the tissue and vascular system leads to the spread of tumor cells into the bloodstream, significantly increasing the level of circulating tumor cells (CTCs). CTCs spread throughout the body via the bloodstream, increasing the incidence of distant metastasis. Therefore, intraoperative bleeding not only increases the postoperative recovery burden on patients but also promotes the metastasis and spread of tumor cells, significantly increasing the risk of postoperative metastasis (Cancer Res. 2017 Apr;77(7):1548-1552).

[0004] Gemcitabine, a first-line chemotherapy drug for pancreatic cancer, is a difluoronucleoside antimetabolite that disrupts cell replication. It was launched in the United States in 1996 and subsequently approved for marketing in over 90 countries worldwide, with global sales reaching $1.7 billion in 2008. However, most clinical chemotherapy strategies involve intravenous infusion, meaning the drug undergoes primary metabolism in the liver after entering the bloodstream, resulting in very low concentrations reaching the surgical site to exert their effects. Electrospun implants can be applied in situ within the surgical cavity, ensuring direct drug delivery to the tumor resection site and significantly reducing the toxicity risks associated with systemic chemotherapy. For example, Wu et al. (Adv Mater. 2023 Dec;35(51):e2305529.) used a roller electrospinning process to fix different polymers layer by layer onto a micromold, creating a layered microdevice. Through in situ injection, they regulated drug release in a mouse model of postoperative pancreatic tumors, significantly increasing drug concentration at the tumor site, reducing systemic toxicity, and effectively inhibiting tumor growth, recurrence, and metastasis. This paper employs roller electrospinning technology, which mainly achieves programmed drug release through structured design, and requires injection for drug delivery.

[0005] Furthermore, the high porosity and high specific surface area of ​​electrospun fibers can provide more opportunities for cancer cells to come into contact, thereby capturing CTCs and reducing the risk of distant metastasis. On the one hand, the high porosity of electrospun sponge structures gives them good air permeability and liquid permeability. The large specific surface area not only facilitates rapid absorption of wound bleeding, but also promotes physical contraction and compression caused by processes such as fiber swelling, thereby improving hemostasis and passively capturing CTC cells in the blood. In addition, in order to enhance the specific capture ability of nanofibers for cancer cells, functionalizing the fiber surface with targeting ligands (such as antibodies, aptamers, avidin, etc.) that can recognize the molecular characteristics of cancer cell surfaces has gradually become a method. For example, Yoon et al. (Nanomedicine. 2017 May;13(5):1617-1625) linked anti-EpCAM monoclonal antibodies to the surface of electrospun polystyrene-poly(styrene-conmaleic anhydride) nanofibers for specific cancer cell isolation. However, the high cost of antibodies and their susceptibility to denaturation and degradation limit their feasibility in widespread application. To overcome these limitations, hyaluronic acid (HA) has emerged as a promising clinical approach due to its high affinity for the CD44 receptor. The CD44 receptor is a cell surface glycoprotein that is expressed at low levels in normal cells but at high levels in various cancer cells, including pancreatic cancer (PharmRes, 2014Nov;31(11):2988-3005). Specific capture of cell-mediated tumors (CTCs) can be achieved by incorporating HA into electrospun nanofiber materials. This HA-CD44 receptor-based targeting strategy provides a new approach for the specific capture and clearance of CTCs after pancreatic cancer surgery (Adv Mater. 2022 Dec;34(49):e2206765).

[0006] Currently, various methods are used to inhibit pancreatic cancer (PC) recurrence, including surgical resection, chemotherapy, and immunotherapy. Postoperative intratumoral drug administration can kill residual pancreatic cancer cells and CTC cells, thereby inhibiting PC growth. Therefore, constructing a delivery system to capture and kill residual pancreatic cancer cells is of great significance for preventing pancreatic cancer recurrence and metastasis. Summary of the Invention

[0007] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing an in-situ "tumor capture" electrospun patch for sequential treatment during and after pancreatic cancer resection.

[0008] Another object of the present invention is to provide a method for preparing the in-situ "tumor capture" type electrospun patch.

[0009] Another object of the present invention is to provide an application of an in-situ "tumor capture" type electrospun patch.

[0010] The objective of this invention can be achieved through the following technical solutions: An in-situ "tumor capture" electrospun patch for sequential treatment during and after pancreatic cancer resection, the electrospun patch comprising a gelatin / hyaluronic acid-based hemostatic sponge layer with capture function, a gemcitabine sustained-release layer, and a polycaprolactone backing layer.

[0011] As a preferred embodiment of the present invention, the mass ratio of gelatin to hyaluronic acid in the gelatin / hyaluronic acid-based hemostatic sponge layer is 20:1 to 80:1, preferably 40:1; the spinning solvent system used in the gelatin / hyaluronic acid-based hemostatic sponge layer is one or more of hexafluoroisopropanol (HFIP), formic acid, water, acetic acid, and N,N-dimethylformamide (DMF), preferably with a volume ratio of HFIP:FA of 7:3.

[0012] As a preferred embodiment of the present invention, the base material used in the gemcitabine sustained-release layer is polylactic acid (PLA) or polylactic acid-glycolic acid copolymer (PLGA), preferably PLA; the mass ratio of gemcitabine to PLA or PLGA is 2:1 to 1:2, preferably 1:1; the spinning solvent system used in the gemcitabine sustained-release layer is HFIP and DMF, preferably with a volume ratio of HFIP to DMF of 9:1.

[0013] As a preferred embodiment of the present invention, the polycaprolactone (PCL) backing layer uses a spinning solvent system of one or more of HFIP, chloroform, methanol, tetrahydrofuran, and DMF, preferably HFIP.

[0014] The electrospun patch of the present invention is prepared by the following method: (1) Prepare drug-loaded spinning solutions containing 15%~30% PLA or PLGA and 15%~30% GEM and 12%~20% PCL respectively. First, electrospin the drug-loaded spinning solution to prepare drug-loaded polylactic acid spinning sheets. Then, electrospin the polycaprolactone spinning solution on the drug-loaded polylactic acid spinning sheets to prepare a bilayer fiber membrane drug-loaded polylactic acid / polycaprolactone bilayer fiber membrane. (2) Prepare a spinning solution containing 15%~30% gelatin and 0.1%~1% hyaluronic acid, spin gelatin / hyaluronic acid spinning sheet, control the thickness within the range of 0.5-1 cm, after electrospinning, weigh the spinning sheet, cut it into pieces and add tert-butanol to disperse it, add steel balls, and homogenize at low temperature until the spinning sheet becomes a paste. (3) The double-layer fiber membrane obtained in step (1) is laid on the bottom of the confocal dish, and the gelatin / hyaluronic acid paste fiber liquid prepared in step (2) is spread in the dish and freeze-dried to obtain a complete electrospun patch.

[0015] As a preferred embodiment of the present invention, in step (1), the volume of the drug-loaded spinning solution and the PCL spinning solution is HFIP:DMF=9:1, and in step (2), the spinning solution containing gelatin and hyaluronic acid is prepared using HFIP:FA=7:3 as the solvent.

[0016] As a preferred embodiment of the present invention, the gelatin / hyaluronic acid-based hemostatic sponge layer has a gelatin mass concentration of 20% and a hyaluronic acid mass concentration of 0.5%; the gemcitabine sustained-release layer has a PLA or PLGA mass concentration of 20% and a GEM mass concentration of 20%; and the polycaprolactone backing layer has a PCL mass concentration of 15%.

[0017] The application of the electrospun patch described in this invention in the preparation of drugs for tumor chemotherapy, radiotherapy, immunotherapy, gene therapy, and wound repair.

[0018] Beneficial effects: The in-situ "tumor capture" electrospun patch constructed in this patent has the following benefits: (1) Immediate hemostasis: It meets the urgent need for rapid hemostasis of postoperative wounds and has a rapid response immediate hemostasis function. This layer structure utilizes the high hydrophilicity of gelatin and the loose and porous structure of sponge, which can effectively absorb exudate and promote the coagulation cascade reaction upon contact with the postoperative wound, achieving rapid wound closure (hemostasis within 30 seconds). It can meet the clinical needs for rapid and reliable hemostasis in surgery, effectively reduce the risk of postoperative blood loss, and at the same time build a stable local environment for the precise delivery and long-term effect of subsequent anticancer drugs.

[0019] (2) Passive and active capture of cancer cells. At the same time, through swelling, the gaps are reduced to form a preliminary capture cage. In addition, hyaluronic acid specifically recognizes CD44 and captures residual circulating tumor cells (the cell capture efficiency reaches more than 90%), blocking hematogenous metastasis caused by surgery.

[0020] (3) Long-acting sustained release and sustained killing: continuous and stable local release of gemcitabine, maintaining long-term anti-cancer activity (release behavior still exists after 14 days).

[0021] This invention integrates the dual core needs of intraoperative and postoperative hemostasis and anti-cancer treatment, and constructs a long-lasting protective barrier at the surgical site through continuous drug delivery. This not only significantly improves the efficiency of removing residual lesions, but also effectively prevents local recurrence and distant metastasis of tumors, ultimately providing pancreatic cancer patients with a safer, more durable, and more comprehensive new treatment strategy. Attached Figure Description

[0022] Figure 1Flowchart for the preparation of in-situ "tumor capture" type electrospun patch; Figure 2 Scanning electron microscope image of a gelatin / hyaluronic acid-based hemostatic sponge layer Figure 3 Scanning electron microscope image of gemcitabine sustained-release layer; Figure 4 Drug release curves for in-situ "tumor capture" type electrospun patches; Figure 5 The water contact angle of the in-situ "tumor capture" type electrospun patch; Figure 6 Evaluation of hemostasis for in-situ "tumor capture" type electrospun patch; Figure 7 Cell compatibility of in situ "tumor capture" type electrospun patches; Figure 8 Evaluation of the capture capability of in-situ "tumor capture" type electrospun patch; Figure 9 Evaluation of the tumor-killing ability of in-situ "tumor capture" type electrospun patch; Figure 10 For the surgical and implantation of in situ "tumor capture" electrospun patch process in mice; Figure 11 Fluorescence monitoring of tumor recurrence in mice after surgery using in situ "tumor capture" type electrospun patches; Figure 12 Quantitative fluorescence analysis of in situ "tumor capture" type electrospun patch in mice after surgery; Figure 13 Evaluation of tumor weight in mice after surgery using in situ "tumor capture" type electrospun patches; Figure 14 Evaluation of the recurrence rate of in situ "tumor capture" type electrospun patch in mice after surgery; Figure 15 Evaluation of the survival of in situ "tumor capture" electrospun patches in mice after surgery. Detailed Implementation

[0023] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0024] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0025] Example 1: Preparation of in-situ "tumor capture" type electrospun patch Polylactic acid (PLA) and gemcitabine (GEM) were placed in vials and mixed with HFIP:DMF (9:1 ratio) to prepare a drug-loaded spinning solution containing 20% ​​(w / v) PLA and 20% (w / v) GEM. The solution was stirred overnight at room temperature. Polylactic acid (PCL) was placed in vials and mixed with HFIP to prepare a 15% (w / v) PCL spinning solution. The solution was stirred overnight at room temperature. The mixed drug-loaded spinning solution was transferred to a 5 ml syringe. A 20 G nozzle was selected, connected to the syringe, and placed in a micro-injector. The positive output of a high-voltage DC power supply was connected to the metal part of the nozzle, and the negative output was connected to the tin foil on the receiving plate. The distance between the receiving plate and the nozzle was adjusted to 18 cm. The voltage of the high-voltage DC power supply was set to 9 kV, and the injection speed of the micro-injector was set to 0.1 mm / min. The electrospinning environment was kept dry at room temperature for 3 h, yielding a drug-loaded polylactic acid spun sheet (2-layer drug-loaded assembly). If only PLA is prepared as a spinning solution according to the above formula (without GEM), and the above process is followed, a drug-free polylactic acid (PLA) spun sheet (2-layer blank group) is prepared. Electrospinning continues on the drug-loaded PLA spun sheet. A 15% (w / v) PCL solution is transferred to a 5 ml syringe, a 20 G nozzle is selected, the spinning distance is adjusted to 18 cm, the voltage is set to 12 kV, the microsyringe advance speed is set to 0.15 mm / min, the electrospinning environment is placed in a dry room temperature, and the electrospinning time is 2 h, thus preparing a drug-loaded PLA / polycaprolactone bilayer fiber membrane (2+3-layer drug-loaded group). If the PCL spinning solution is spun on the surface of the drug-free PLA spun sheet, a... Drug-free polylactic acid / polycaprolactone bilayer fiber membrane (2+3 layer blank group). If PCL is spun onto the receiving plate using the above process, a polycaprolactone PCL backing layer (3-layer backing group) is prepared.

[0026] Gelatin GE (20%, w / v) and hyaluronic acid HA (0.5%, w / v) were dissolved in a solvent system of HFIP:FA = 7:3 (v / v) and stirred at 40°C for 4 h to obtain a spinning solution. The spinning solution was transferred to a 5 mL syringe with a 20 G nozzle, and the injection speed of the microsyringe was set to 0.2 mm / min. The needle tip was 18 cm away from the receiving plate, and the voltage of the high-voltage DC power supply was set to 15 kV. The thickness of the gelatin / hyaluronic acid spun sheet was controlled within the range of 0.5-1 cm. After electrospinning, 50 mg of the spun sheet was weighed, cut into small pieces, and 2 ml of tert-butanol was added to disperse it. The mixture was then homogenized in a cryogenic homogenizer at 3000 rpm for 1 min, and repeated five times. At this point, the spun sheet should be a homogeneous white paste-like liquid.

[0027] The prepared drug-loaded polylactic acid / polycaprolactone bilayer fiber membrane was laid on the bottom of a confocal microscopy dish (with the polycaprolactone layer at the bottom). The gelatin / hyaluronic acid paste-like fiber solution prepared in the previous step was then spread evenly in the dish, and freeze-dried to obtain an in-situ "tumor capture" type electrospun patch (drug-loaded complete sponge formulation). If the GE / HA blended spinning solution was replaced with the same GE single-component spinning solution, the in-situ "tumor capture" type electrospun patch prepared according to the same process was a drug-loaded complete sponge formulation (without HA). If the GE / HA blended spinning solution was directly freeze-dried in the dish according to the same process without adding the drug-loaded polylactic acid / polycaprolactone bilayer fiber membrane, a gelatin / hyaluronic acid fiber sponge (single-layer sponge group) was prepared. If the GE / HA blend is directly spun onto the surface of a drug-loaded polylactic acid / polycaprolactone bilayer fiber membrane, using a 20 G nozzle, a microsyringe with a feed rate of 0.2 mm / min, a needle tip distance of 18 cm from the receiving plate, and a high-voltage DC power supply of 15 kV, spinning for 1 hour yields a drug-loaded complete membrane formulation. Alternatively, if the prepared unloaded polylactic acid / polycaprolactone bilayer fiber membrane is laid on the bottom of a confocal dish (with the polycaprolactone layer at the bottom), and the gelatin / hyaluronic acid paste-like fiber solution prepared in the previous step is spread evenly in the dish, and then freeze-dried, a blank complete sponge formulation is obtained.

[0028] Table 1

[0029] Example 2: Characterization and performance testing of in-situ "tumor capture" type electrospun patch (1) Morphological investigation of the first layer of sponge The morphology of the single-layer sponge assembly prepared in Example 1 was observed using scanning electron microscopy. The specific procedures were as follows: the prepared single-layer sponge assembly was immersed in liquid nitrogen, frozen for 2 minutes, and then fractured. A cross-section was sputter-coated with gold for 60 seconds to obtain the SEM sample. Finally, it was fixed to the sample stage using conductive adhesive for observation and imaging. Figure 2 As shown, a loose honeycomb nanofiber network was successfully obtained after homogenization foaming treatment. The porous structure exhibited by the sponge group facilitates the capture of tumor cells and the absorption of blood.

[0030] (2) Morphological examination of the two-layer drug-loaded group and the two-layer blank group The morphology of the two-layer drug-loaded group and the two-layer blank group prepared in Example 1 were observed using scanning electron microscopy at a voltage of 3 kV. The specific operating steps are as follows: each group of electrospun fiber sheets was cut into standard sizes of 1 cm × 1 cm, then coated with gold for 60 s to prepare the sample to be tested. Finally, it was fixed on the sample stage using conductive adhesive and observed by SEM. The fibers in the spun sheets were smooth strips with good uniformity. Through comparison... Figure 3 (a) shows the 2-layer blank group and Figure 3 (b) The graph of the two-layer drug-loaded group shows that gemcitabine is evenly distributed in the fiber, without any protrusions on the surface, and does not affect the fiber morphology.

[0031] (3) Investigation of drug release behavior Weigh out 2 mg of the in-situ "tumor capture" electrospun patch (drug-loaded intact sponge formulation) containing GEM prepared in Example 1 and incubate it at 37°C in 20 mL of phosphate-buffered saline (0.01 mM, pH=7.4) with gentle shaking (100 rpm). At predetermined time intervals, collect 0.5 mL of release medium and add the same volume of fresh medium. Determine the peak value and elution time of gemcitabine hydrochloride, measure the corresponding peak area, and calculate the concentration. Figure 4 As shown, gemcitabine release is stable, with a release rate of approximately 72% in the first 14 days. The sustained-release effect of GEM in the in-situ "tumor capture" electrospun patch depends on the PLA encapsulation. On the other hand, the chelate interaction between the sponge layer and the gelatin carboxyl groups and gemcitabine may further limit the rapid release of gemcitabine from the fiber, thus prolonging the release time and rate of gemcitabine.

[0032] (4) Investigation of water affinity A one-layer sponge group, a two-layer drug-loaded group, and a three-layer backing group were prepared according to the method in Example 1, and the hydrophobicity of each layer was evaluated using a contact angle meter. Figure 5 As shown, 2 μL of deionized water was added to the sample surface, and the droplet morphology was captured after standing for 60 seconds. The static contact angle was calculated using the Young-La equation. Three regions were measured for each layer, and the average value was taken. The hydrophobicity gradient of each layer was determined based on the contact angle: 3-layer backing group > 2-layer drug-loaded group > 1-layer sponge group.

[0033] (5) Evaluation of hemostatic performance To evaluate the hemostatic performance of the in-situ "tumor capture" electrospun patch, the untreated group, the commercially available gelatin sponge-treated group, and the drug-loaded intact sponge formulation-treated group (the in-situ "tumor capture" electrospun patch prepared in Example 1) were cut into equal volumes and placed in petri dishes. 100 µL of whole blood containing EDTA-2Na (0.08 mg) was dropped onto the surface of each sample. In the control group, the same amount of blood was dropped directly onto the petri dish. All petri dishes were incubated at 37°C for 5 min. Then, 50 mL of distilled water was carefully added to the petri dishes.

[0034] When no clot has formed, red blood cells are more likely to diffuse out of the network and rupture, causing hemolysis. However, after clotting, red blood cells within the clot are less prone to hemolysis. Therefore, the presence of hemolysis can be used to assess the clotting process. Figure 6The color of deionized water in the drug-loaded intact sponge formulation changed slightly, and the hemolysis was weak, proving that blood could automatically coagulate on the electrospun patch within 5 minutes, and the hemostatic ability was better than that of commercially available sponges and the untreated group.

[0035] (6) Compatibility evaluation NIH / 3T3 mouse embryonic fibroblasts were used at a dose of 1×10 5 Cells were seeded at the specified well density in 24-well plates. One layer of sponge, two layers of blank, three layers of backing, and a blank intact sponge preparation were sterilized by UV irradiation for 1 hour and then placed in the wells. Cell suspension containing 0.1 g / mL DMEM culture medium was added. A control group without material was included. Cell viability was quantitatively analyzed using the MTT assay after 24 hours.

[0036] like Figure 7 As shown, the cell survival rates of the 1-layer sponge group, the 2-layer blank group, and the blank intact sponge preparation group were all above 98%, maintaining a high level, indicating that the blank preparation had no obvious toxicity to cells and had high biocompatibility. However, due to the anti-adhesion properties of the material, cell adhesion and proliferation were significantly restricted in the 3-layer backing group, resulting in a lower survival rate compared to the other groups, which was in line with expectations.

[0037] (7) Evaluation of tumor capture capability Pancreatic cancer cells from PAN02 mice and embryonic fibroblasts from NIH / 3T3 mice were injected at a dose of 1×10⁻⁶. 5 The cells were seeded at a density of 10 µg / mL in 24-well plates, Hoechst staining solution was added, and the plates were stained at room temperature for 10 min. After removing the staining solution, the cells were washed three times with PBS, digested, centrifuged, and collected. Then, PAN02 and NIH / 3T3 cells were added to 24-well plates with two blank layers, blank intact membrane preparation, blank intact sponge preparation (without HA), and blank intact sponge preparation as substrates, respectively. The control group was not added to any preparation. After incubation at 37°C for 40 min, the substrates were gently washed three times with PBS. The group without preparation and without washing was used as a positive control.

[0038] like Figure 8 Under both blank intact sponge formulation (without HA) and blank intact sponge formulation conditions, the cell numbers in both the PAN02 and 3T3 groups increased significantly, indicating that the fluffy sponge structure has excellent cell capture ability. Under the blank intact sponge formulation conditions, the number of cells captured by PAN02 further increased compared to the HA-free group. This is due to the specific binding of hyaluronic acid to the transmembrane protein CD44 on the PAN02 surface, which further promotes the capture of tumor cells by the formulation.

[0039] (8) Evaluation of tumor killing ability Blank and drug-loaded intact sponge formulations were cut into small pieces and irradiated with ultraviolet light for approximately 1 hour for cell detection. PAN02 cells were cultured at a rate of 2 × 10⁻⁶. 4 Cells were seeded at a density of [insert density here] in 96-well plates and incubated overnight. The old culture medium was discarded, and three replicates were made for each group. 100 μL of DMEM, blank intact sponge formulation, and drug-loaded intact sponge formulation (100 μM GEM) were added to each well. An equal volume of DMEM served as a negative control, and DMEM containing 100 μM free GEM served as a positive control. The plates were incubated at 37°C with 5% CO2 for 24 h. Live and dead cells were stained with AM / PI dyes. Cell morphology was observed using an inverted fluorescence microscope / confocal microscope.

[0040] Figure 9 Cells in the blank intact sponge formulation showed almost no cell death. The number of dead cells in the drug-loaded formulation group was comparable to that in the free GEM group, and significantly higher than that in the blank formulation group. These results indicate that the drug-loaded intact sponge formulation with GEM has an inhibitory effect on tumor cells.

[0041] Example 3: In vivo pharmacodynamic evaluation of in situ "tumor capture" type electrospun patch (1) Mouse surgery and in situ "tumor capture" type electrospun patch implantation process Male C57BL / 6 mice were randomly divided into 3 groups. After anesthesia, the skin and external smooth muscle of the spleen were incised, with an incision of approximately 1.0 cm. The tail of the pancreas was located using a sterile moistened cotton swab, and approximately 50 μL of 1×10⁻⁶ pancreas solution was suspended. 6 Pan02-Luc cells were injected into the tail of the pancreas using DMEM / Matrigel substrate (1:1, v:v), and the abdominal wall and skin wound were sutured. On day 21 post-surgery (fluorescence intensity greater than 10), the cells were observed. 7 p / s / cm 2 After ( / sr), mice were anesthetized, and abdominal incision was performed to prepare the tumor-bearing pancreatic tail. Blood vessels within the spleen were ligated with sutures, and the incision surface was cut with scissors. Careful management of the resection margins and residual tissue was maintained to ensure incomplete tumor removal, leaving approximately 10% residual tumor to simulate postoperative microtumor remnants. Different treatments were administered to each group: ① saline treatment ② local implantation of a single layer of sponge (1 cm × 1 cm) ③ drug-loaded intact sponge preparation (1 cm × 1 cm). Figure 10 As shown, the process of tumor exposure, resection, drug implantation, hemostasis, segmentation, and other procedures were documented by photographs during the surgery.

[0042] (2) Tumor fluorescence intensity monitoring D-fluorescein sodium was injected intraperitoneally once a day after administration, and the fluorescence intensity of the tumor site was recorded using a small animal in vivo imaging system. Figure 11The study demonstrated the monitoring of tumor progression in mice after surgery using in-situ "tumor capture" electrospun patches. The results showed that the drug-loaded intact sponge formulation significantly reduced postoperative tumor progression in mice.

[0043] (3) Quantitative monitoring of tumor fluorescence intensity Automated quantitative analysis of the region of interest (ROI) was performed on the fluorescence intensity of tumor sites recorded by a small animal in vivo imaging system. Figure 12 The fluorescence intensity of mice in the drug-loaded intact sponge formulation group showed a decreasing trend, significantly lower than that in the saline group.

[0044] (4) Tumor weight assessment Twenty-one days after the operation, the mice were dissected, and the tumor tissue was separated and weighed. Figure 13 The study showed the weight of solid tumors in mice after surgery using in-situ "tumor capture" electrospun patches. The results showed that the drug-loaded intact sponge formulation significantly inhibited tumor growth in mice after surgery, with a weight lower than that of the saline group and the blank formulation group.

[0045] (5) Investigation of postoperative recurrence rate in mice Relapse in mice was monitored weekly using quantitative fluorescence intensity data (fluorescence intensity greater than 1×10⁻⁶). 7 p / s / cm 2 / sr, then it is considered a relapse). Figure 14 The results showed that the saline group had relapsed by the fourth day, while the drug-loaded intact sponge preparation group had a relapse rate of less than 40% (three mice never relapsed), demonstrating the effectiveness of the preparation in preventing relapse.

[0046] (6) Postoperative survival of mice Day 0 was recorded as the start of postoperative drug administration. The survival time and recurrence rate of mice in each group were observed, and survival curves were plotted. Figure 15 The study showed that the drug-loaded intact sponge formulation significantly improved the postoperative survival of mice, with a survival rate of 60% on day 40 (3 mice survived).

Claims

1. An in-situ "tumor capture" type electrospun patch for sequential treatment during and after pancreatic cancer resection, characterized in that, The electrospun patch includes a gelatin / hyaluronic acid-based hemostatic sponge layer with capture function, a gemcitabine sustained-release layer, and a polycaprolactone backing layer.

2. The electrospun patch according to claim 1, characterized in that, The mass ratio of gelatin to hyaluronic acid in the gelatin / hyaluronic acid-based hemostatic sponge layer is 20:1 to 80:1, preferably 40:1; the spinning solvent system used in the gelatin / hyaluronic acid-based hemostatic sponge layer is one or more of hexafluoroisopropanol (HFIP), formic acid, water, acetic acid, and N,N-dimethylformamide (DMF).

3. The electrospun patch according to claim 1, characterized in that, The volume ratio of HFIP to FA is 7:

3.

4. The electrospun patch according to claim 1, characterized in that, The gemcitabine sustained-release layer uses polylactic acid (PLA) or polylactic acid-glycolic acid copolymer (PLGA) as the base material, with a gemcitabine to PLA or PLGA mass ratio of 2:1 to 1:2; the gemcitabine sustained-release layer uses HFIP and DMF as the spinning solvent system.

5. The electrospun patch according to claim 1, characterized in that, The gemcitabine sustained-release layer uses polylactic acid (PLA) as its base material; the mass ratio of gemcitabine to PLA or PLGA is 1:1; the spinning solvent system used in the gemcitabine sustained-release layer is a solvent with a volume ratio of HFIP to DMF of 9:

1.

6. The electrospun patch according to claim 1, characterized in that, The polycaprolactone (PCL) backing layer uses a spinning solvent system of one or more of HFIP, chloroform, methanol, tetrahydrofuran, and DMF, preferably HFIP.

7. The electrospun patch according to any one of claims 1-6, characterized in that, It is prepared by the following method: (1) Prepare a mixed drug-loaded spinning solution containing 15%~30% PLA or PLGA and 15%~30% GEM and a 12%~20% PCL spinning solution respectively. First, electrospin the mixed drug-loaded spinning solution to prepare a drug-loaded polylactic acid spinning sheet. Then, electrospin the polycaprolactone spinning solution on the drug-loaded polylactic acid spinning sheet to prepare a bilayer fiber membrane drug-loaded polylactic acid / polycaprolactone bilayer fiber membrane. (2) Prepare a spinning solution containing 15%~30% gelatin and 0.1%~1% hyaluronic acid, spin gelatin / hyaluronic acid spinning sheet, control the thickness within the range of 0.5-1 cm, after electrospinning, weigh the spinning sheet, cut it into pieces and add tert-butanol to disperse it, add steel balls, and homogenize at low temperature until the spinning sheet becomes a paste. (3) The double-layer fiber membrane obtained in step (1) is laid on the bottom of the confocal dish, and the gelatin / hyaluronic acid paste fiber liquid prepared in step (2) is spread in the dish and freeze-dried to obtain a complete electrospun patch.

8. The electrospun patch according to claim 8, characterized in that, In step (1), the volume of the drug-loaded spinning solution and the PCL spinning solution is HFIP:DMF=9:1, and in step (2), the spinning solution containing gelatin and hyaluronic acid is prepared using HFIP:FA=7:3 as the solvent.

9. The electrospun patch according to claim 8, characterized in that, The gelatin / hyaluronic acid-based hemostatic sponge layer has a gelatin mass concentration of 20% and a hyaluronic acid mass concentration of 0.5%; the gemcitabine sustained-release layer has a PLA or PLGA mass concentration of 20% and a gemcitabine mass concentration of 20%; and the polycaprolactone backing layer has a PCL mass concentration of 15%.

10. The use of the electrospun patch according to any one of claims 1-6 in the preparation of drugs for tumor chemotherapy, radiotherapy, immunotherapy, gene therapy, and wound repair.

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